The goal of this research is to develop an economical, safe, effective therapy to reverse the activity of the catalytic component, LF, of the Bacillus anthracis toxin (lethal toxin), within the cytoplasm of the intoxicated cell. Success in achieving this goal will lead to better outcomes than currently seen with antibiotics or antitoxins like raxibacumab or PBI-220, and this could save countless more lives in the event of a bioterrorist attack with B. anthracis. We propose to screen the humanized camelid library to identify high-affinity single chain variable fragment, a nanobody, able to block protease activity of lethal factor (LF). We will then create genetic fusion of the nanobody that blocks LF with another domain of LF, LFN, that had the translocating activity buts is devoid of catalytic activity to enable it to be transported into the cytoplasm of mammalian cells in the presence of PA. Thus, we plan to co-opt the B. anthracis toxin delivery system to deliver, instead, a neutralizing nanobody into intoxicated cells. This project involves two research teams at two institutes, Panaorama Research Institute (PRI) and Planet Biotechnology (PBI). At PRI, biotinylated LF will be used to screen the humanized camelid library and identify clones that have high affinity and are ability to block protease activty of LF. They will also be selected to ensure that they do not bind LFN, the domain that will be used to translocate them into the cell. We will modify the nanobody to make fusion proteins with the LFN domain, giving it the ability to be translocated into cells, in a PA-dependent manner, and be able to reach LF in the cytoplasm of intoxicated cells. We will express the nanobody fusion proteins using the rapid transient plant expression system and purify the fusions using affinity chromatography. First, we will test their ability to enter cells and subsequently measure their ability to inhibit LF enzymatic activity in intoxicated cells.